J. Nannoplankton Res. 30 (2), 2009, pp.77-80 © 2009 International Nannoplankton Association, Inc.
ISSN 1210-8049 Printed by Cambridge University Press, UK

77

Effect of elevated nitrate concentration on calcification inEmiliania huxleyi
Gerald Langer
Alfred Wegener Institute for Polar and Marine Research, 27570 Bremerhaven, Germany & Institute of Environmental Science and Technology(ICTA), Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Spain; Gerald.Langer@awi.deIna Benner
Alfred Wegener Institute for Polar and Marine Research, 27570 Bremerhaven, Germany

Manuscript received 24th April, 2008; revised manuscript accepted 11th December, 2008

Abstract A monoclonal culture of Emiliania huxleyi (PML92/11) was grown in natural sea-water under varying
nitrate concentrations, ranging from 10 to 890μmol L-1. Growth rate, calcification rate, particulate organic carbon production, particulate organic nitrogen production, and the ratio of aberrant coccoliths to normal coccoliths were determined. None of these parameters showed a trend over the range of nitrate concentrations tested. It is concluded thathigh nitrate concentrations, typical for culture media like f/2, do not affect morphogenesis of coccoliths, nor do theyaffect the production of particulate organic carbon or nitrogen in this strain.

Keywords Emiliania huxleyi culture, nitrate concentration, growth rate, calcification, particulate organic carbon,
particulate organic nitrogen, coccolith malformation

1. Intoduction
Coccolithophores are unicellular marine algae, which haveevolved the ability to produce minute calcite platelets, thecoccoliths. From a biogeochemical point of view, coccolithophores are of special interest, since, besides being important primary producers, they are responsible forapproximately half of modern marine pelagic calcium carbonate export production (Baumann et al., 2004). The biogeochemical significance of coccolithophores has
motivated a number of studies concerned with different aspects of the biogeochemistry, cell biology and physiologyof these algae. Besides issues that can be dealt with in fieldstudies, numerous questions can only be answered bymeans of controlled laboratory experiments using monoclonal cultures. Therefore, the significance of culturebanks, such as the Roscoff Culture Collection (www.sbroscoff.fr/Phyto/RCC), must not be underestimated.
Despite the noted success of standard culturing techniques (Probert & Houdan, 2004), it is well known that thepercentage of aberrant coccoliths in cultures is higher thanin natural samples (Langer et al., 2006). The reason for thishampered morphogenesis of coccoliths in cultured specimens is unknown. Since calcification-related data, such ascalcification rate and coccolith morphology, play a majorrole in coccolithophore research, there is, on one hand, aneed to solve this riddle. On the other hand, there are also
many possible answers, which is presumably the reason
why systematic studies concerned with this question arelacking. Culturing of marine microalgae traditionally includes the usage of sea-water-based media and special additives, including micro- and macronutrients (Probert &Houdan, 2004). For instance, the nitrate concentration of
the often-used f/2 medium (Guillard, 1975) is 880μmol
L-1, whereas typical nitrate concentrations in the North At-

lantic range from 0.2μmol L-1 to 30μmol L-1 (Garcia et al.,2005). 14C uptake experiments, using the most-studied coccolithophore Emiliania huxleyi, have revealed a decreased
calcification rate under 1000μmol L-1 nitrate (Nimer &Merrett, 1993). Since calcification rate and coccolith morphology were shown to be altered in concert by environmental conditions (e.g. Langer et al., 2006), it is also likelythat morphogenesis of E. huxleyi coccoliths is impaired byf/2 nitrate concentration. We have addressed this question
by growing a monoclonal culture of E. huxleyi in naturalsea-water enriched with different nitrate concentrations.
Calcification rate, coccolith morphology, growth rate, particulate organic carbon production and particulate organic
nitrogen production were determined.
2. Material and methods
A monospecific culture of Emiliania huxleyi (strainPML92/11) was grown in sterile filtered (0.2μm) sea-water
enriched with 7μM phosphate, and with trace metals andvitamins according to f/2 (Guillard, 1975). Sea-water wascollected from surface waters off Helgoland (Germany).
Nutrient samples (30mL) were filtered throughprecombusted (12 hours, 500˚C) glass-fibre filters(Whatman GF/F), and nitrate plus nitrite (NOx) wasmeasured on an Alliance EVOLUTION3 Autoanalyser,according to Hansen & Koroleff (1999). The sea-water off
Helgoland had a natural NOx concentration of 10.3μmolL-1 (which is considered as nitrate, because the percentage
of nitrite is usually negligible). Cells were grown in seawater with the following nitrate concentrations: 10μmolL-1, natural sea-water without nitrate added (NSW),
98μmol L-1, NSW with 88μmol L-1 nitrate added (f/20),
230μmol L-1, NSW with 220μmol L-1 nitrate added (f/8),450μmol L-1, NSW with 440μmol L-1 nitrate added (f/4),

Langer, Benner78

and 890μmol L-1, NSW with 880μmol L-1 nitrate added(f/2). The incident photon flux density was 270μmol m-2
s-1 and a 16/8-hour light/dark cycle was applied.
Experiments were carried out at 17˚C. Cells wereacclimated to experimental conditions for approximately
10 generations and grown in dilute batch cultures, induplicate (Langer et al., 2006).
Dissolved inorganic carbon (DIC) samples were sterilefiltered (0.2μm) and stored in 13mL borosilicate flasks, freeof air bubbles, at 0˚C. DIC was measured photometrically(Stoll et al., 2001), in triplicate. Less than 4% DIC
consumption (i.e. DIC consumed by the cells at the end ofthe experiment) ensured insignificant changes in carbonate
chemistry during growth (Langer et al., 2006). Samples fordetermination of total particulate carbon (TPC) andparticulate organic carbon (POC) were filtered on
precombusted (12 hours, 500˚C) GF/F filters (Whatman,approx. 0.7μm) and stored at -20˚C. Prior to analysis,
230μL of a HCl solution (5mol L-1) was added on top of
the POC filters to remove all inorganic carbon. TPC andPOC were subsequently measured on a Euro EA Analyser
by Euro Vector. Particulate inorganic carbon (PIC) wascalculated as the difference between TPC and POC.
For determination of cell density, samples were taken
daily, and counted directly after sampling using a Coulter
Multisizer III. Cell density was plotted against time, andgrowth rate (μ) was calculated by means of exponential
regression and is given in [per day]. Particulate inorganiccarbon production (PIC production) was calculated
according to:
P (PIC) = μ * (cellular inorganic carbon content) [pg PIC cell-1 day-1]

Particulate organic carbon production (POC production)
was calculated according to:
P (POC) = μ * (cellular organic carbon content) [pg POC cell-1 day-1]

Particulate organic nitrogen production (PON production)
was calculated according to:
P (PON) = μ * (cellular organic nitrogen content) [pg PON cell-1 day-1]

Particulate inorganic carbon production is termed‘calcification rate’ in the following.
Samples for scanning electron microscope (SEM)
analysis were filtered on polycarbonate filters (0.2μm poresize), dried in a drying cabinet at 60˚C for 24 hours, thensputter-coated with gold-palladium. Imaging wasperformed with a Philips XL-30 digital scanning fieldemission electron microscope. Three categories were used
to describe the morphology of E. huxleyi coccoliths:‘normal’, ‘malformed’ and ‘incomplete’ (for reference
images for the categories, see Figure 1). An average of~350 coccoliths was analysed per sample.

3. Results and discussion
The coccolith morphology of Emiliania huxleyi, grown innatural sea-water with elevated nitrate concentration, was analysed.
To quantify morphology, coccoliths
were divided into three categories,
namely ‘normal’, ‘malformed’ and‘incomplete’ (Figure 1). The percentages of coccoliths in the threecategories did not change significantly with nitrate concentration(Figure 2, Table 1). This shows
clearly that the unnaturally high nitrate concentration of the common
sea-water supplement f/2 (Guillard,2μm1μm1975) does not influence coccolithmorphology. The percentage of malformed or incomplete coccoliths
was ~30% (Figure 2, Table 1). In
oceanic samples, this number is, typically, one order of magnitude
smaller. However, in the stock culture of the strain used in this study,
we have occasionally observed up to98% of malformed or incomplete
2μmcoccoliths (cells grown in the f/21μmmedium). The reason for the highlyvariable degree of malformation in
Figure 1: SEM images of Emiliania huxleyi coccoliths. A) Two normal coccoliths, showing proxicoccoliths of cultured specimensmal (background) and distal (foreground) shields. B) Two incomplete coccoliths, comprising prototherefore remains obscure.
coccolith ring and incipient central area. C) Incomplete coccolith, at ~80% growth. D) Two malformed
Calcification rate did notcoccoliths, showing distal (left) and proximal (right) shields

ABCD

Effect of elevated nitrate on...E. huxleyi

Figure 2: Percentage of normal, malformed and incomplete coccoliths vs.
nitrate concentration. Values represent an average of duplicate experimentschange with increasing nitrate concentration either (Figure3, Table 2). However, in an earlier study, decreased calcification rate of E. huxleyi under 1000μmol L-1 nitrate has
been observed (Nimer & Merrett, 1993). The apparent discrepancy between our results and the results of Nimer &Merrett (1993) can be reconciled: firstly, there might bestrain-specific responses involved - Nimer & Merrett(1993) used a different strain; and, secondly, the methodologies of the two approaches are quite different. Calcification rate, as measured in this study, is an integrated value
over the course of the experiment, that is, over several generations and including light and dark phases (see Materialand methods, above). Nimer & Merrett (1993) conductedshort-term (minutes) 14C-incorporation measurements,
which do not capture lag phases between coccolith production. This latter study, however, showed that there isprincipally some mechanism by which the nitrate concentration typical for the f/2 medium can affect the process ofcalcification.
In another study using E. huxleyi (strain 92-a), it wasobserved that cells produced no coccoliths under 1800μmol

79

L-1 nitrate, whereas they did under 200μmol L-1 (Wilbur &Watabe, 1963). Another strain of E. huxleyi (strain BT-6),
however, developed coccoliths at both 200μmol L-1 and
1800μmol L-1 (Wilbur & Watabe, 1963). This observationsuggests that strain-specific effects play a considerable rolein the physiological response to high nitrate concentrations.
However, we did not test 1800μmol L-1, because there isno common sea-water-supplement recipe including such ahigh nitrate concentration (Probert & Houdan, 2004).
Hymenomonas sp. was grown under even higher nitrateconcentrations, namely 6mmol L-1 and 24mmol L-1 (Baumann et al., 1978). A decrease in the particulate inorganic
calcium to particulate nitrogen ratio was observed with increasing nitrate concentration, indicating either a hampered
calcification or an increased nitrogen assimilation underhigh nitrate concentration. Since the authors reported neither particulate calcium nor particulate nitrogen normalised
to cell nor growth rates, the two processes, that is, calcification and nitrogen assimilation, cannot be distinguished.
For E. huxleyi, we measured growth rate, cellular PIC, POC

Figure 3: Calcification rate vs. nitrate concentration. Values represent anaverage of duplicate experiments

and PON content, and PIC, POC and PON production (Figures 3 and 4, Table 2). None of these parameters showed atrend over the range of nitrogen concentrations tested.
The difference compared with the results of Baumann
nitrate concentration normal % malformed % incomplete %et al. (1978) might be explained by either speciesspecific effects or the higher nitrate concentrations
NSW A
82
108
tested by Baumann et al. (1978). The fact that POC
NSW B
71
1515
production did not change (Figure 4A, Table 2) is in
f/20 A
64
1818
apparent contradiction to the findings of Nimer &f/20 B
63
1918
Merrett (1993), who described diminished 14C uptake
f/8 A
77
1410rates, with respect to organic carbon. This apparent
f/8 B
73
1215
contradiction can be reconciled by applying the same
f/4 A
64
25
11
reasoning as we did with respect to calcification ratef/4 B
67
2013(see above).
f/2 A
72
21
7To summarise, we have shown that the nitrate
f/2 B
77
158
concentration of f/2 affects neither organic carbon or
Table 1: Percentages of normal vs. malformed vs. incomplete coccoliths derived
organic nitrogen production, nor calcification rate orfrom the morphology analysis. Data from duplicate experiments (A, B) listed
coccolith morphology of E. huxleyi (PML92/11). It
separately

Langer, Benner

80

nitrate concentration
PIC content
POC content
PON content
PIC production
POC production
PON production
growth rate μPIC/POC
PIC/PON

pg PIC*cell-1
st. dev.
pg POC*cell-1
st. dev.
pg PON*cell-1
st. dev.
pg PIC*cell-1*d-1
st. dev.
pg POC*cell-1*d-1
st. dev.
pg PON*cell-1*d-1
st. dev.
d-1
st. dev.
mol*mol-1
st. dev.
mol*mol-1
st. dev.

NSW

f/20

f/8

f/4

f/2

7.5
0.4
10.5
0.2
1.7
0.1
10.6
0.6
14.9
0.3
2.3
0.1
1.41
0.05
0.7
0.0
5.1
0.4

8.1
0.2
11.3
0.2
2.0
0.2
11.7
0.3
16.2
0.3
2.8
0.1
1.44
0.02
0.7
0.0
4.7
0.4

8.8
0.3
10.8
0.1
1.6
0.1
12.7
0.4
15.7
0.2
2.3
0.1
1.45
0.00
0.8
0.0
6.2
0.4

8.0
0.3
10.2
0.1
1.6
0.1
11.6
0.5
14.8
0.2
2.2
0.0
1.45
0.01
0.8
0.0
5.8
0.3

8.9
0.3
11.0
0.3
1.8
0.1
12.6
0.4
15.6
0.5
2.4
0.1
1.41
0.00
0.8
0.0
5.8
0.3

remains to be tested whether
this result holds true for otherclones of E. huxleyi, or evenother species.
Acknowledgements
We thank Dr. Paul Bown (UCL)
and an anonymous reviewer for
helpful comments. We are grateful to Kerstin Oetjen, Tina Brenneis and Anja Terbrüggen forlaboratory assistance and fruitful
discussions. GL acknowledges financial support by the Spanish
Ministerio de Educacion y Ciencia through the Juan de la Cierva
program.

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Figure 4: POC production (A) and PON production (B) vs. nitrateconcentration. Values represent an average of duplicate experiments